This invention relates to an apparatus and method for analyzing filter-bound samples and, more particularly, to a low volume perfusion slit chamber for treating and/or developing nucleotide and protein-bound membranes.
Many current methodologies for the high resolution analyses of proteins, nucleic acids, and polynucleotides immobilize samples on thin filter membranes and subsequently analyze the filters using immunologic or recombinant hybridization technologies.
Several years ago, it was found that singlestranded DNA binds very tightly to membrane filters made of nitrocellulose, and that such filters failed to bind either double-stranded DNA or RNA. This phenomenon can be used, for example, to determine the presence of specific complementary base sequences in single-stranded DNA by binding one type of single-stranded DNA to a nitrocellulose filter and reacting the bound single-stranded DNA with labeled, singlestranded DNA or RNA. Where such base sequences are present, the second type of single-stranded DNA or of RNA binds to the filter-bound single-stranded DNA. The presence of double-stranded DNA or a DNA-RNA complex bound to the nitrocellulose sheet can be detected utilizing the label attached to the second type of single-stranded DNA or RNA. If, for example, a radioactive label is used, the presence of a double-stranded DNA or DNA-RNA complex can be detected by exposing x-ray films with the nitrocellulose sheet and developing the x-ray films.
A wide variety of analytical techniques have since been developed utilizing this basic phenomenon. See, e.g., D. Freifelder, Molecular Biology, pp. 123-124 and 418-425 (1983). For example, when single-stranded polynucleotides (DNA or RNA) are separated electrophoretically in polyacrylamide gels or other media, the separated polynucleotides and polynucleotide fragments can be transferred or "blotted" from the polyacrylamide gels onto nitrocellulose membranes or sheets. The separated single-stranded DNA, bound to the sheet, can now be localized by reacting the bound single-stranded DNA with specific sequences or fragments of single-stranded DNA or RNA that have previously been labeled for detection. Possible labels include fluorescent and other chemical labels, as well as radioactive labels, such as .sup.32 P-phosphate labels and .sup.14 C labels.
Similar technologies have been developed for the localization of electrophoretically separated proteins and/or detection of proteins or samples of genetic material dotted onto filter membranes for screening.
Such methods all require rigid control of reaction temperatures at each step and accurately controlled development times. Because of the complex kinetics of chemical interaction involved, as well as the fact that many of the reagents, such as radioactively labled polynucleotides, are rare and/or difficult to isolate or produce in large quantities, the development reactions must be carried out in minimum effective volumes. This, in turn, requires optimization of the efficiency of the interaction between the filter-bound sample and reagents. In addition, a wide range of reagent types and conditions are routinely used. Often, the filters are variable in size, shape, and type, are fragile, and are difficult to handle.
Filter analyses are usually done (with agitation) in open, large-volume containers or, in the case of nucleic acid analyses, in crude, heat-sealed plastic bags. For most purposes, such arrangements are cumbersome and wasteful of reagents, and are inefficient in terms of both the amounts of reagents required and in the time required to complete a particular reaction. Where the labels used include high specific activity radioisotopes, such systems may also be very hazardous. The operator may be exposed to significant radiation while handling such equipment, which is prone to leakage and spillage of radioactive solutions. It is also difficult, at best, to properly dispose of all radioactive materials used in such systems due to spillage, inconvenience of the operations which must be performed, and the relatively large quantities of radioactive material which must be handled and disposed of; such systems thus also are a potential source of environmental hazard.
The plastic bags used frequently in nucleic acid hybridizations are difficult to work with, are prone to leakage, and require agitation to maximize filter/reagent interactions. Such bags are not reusable, and do not afford any significant protection to the often fragile filters being analyzed. Due to the flexibility of the plastic bags, reagent distribution is often uneven, since the positions of the filters within such plastic bags are not readily controllable. Further, such plastic bags systems require reopening and resealing after every reagent change and are effectively useless for washing steps which require large volumes of wash reagents and agitation of the filters and reagents. While such plastic bag systems allow the exclusion of air bubbles from the filter/reagent interface, and complete immersion of the filters with a relatively small volume of reagent, both plastic bag development systems and development systems utilizing large-volume containers have proven inconvenient, inefficient, non-reproducible, and often unsatisfactory for routine use in the analysis of filter-bound samples.